PROJECT SUMMARY The process through which the two-subunit ribosome assembles at the start codon of an mRNA to initiate protein synthesis is one of the most fundamental and highly regulated steps of gene expression. As such, initiation serves as the target of numerous small-molecule antibiotics and cellular pathogens. Moreover, deregulation of initiation is causally linked to viral infections and tumorigenesis in humans. Given all of this, studies of the molecular mechanism of initiation hold great promise for the identification and characterization of mechanistic steps that can serve as targets for the development of next-generation antibiotics and other small- molecule, anti-viral, and anti-cancer drugs that act by modulating translation initiation. Despite their promise for molecular medicine, mechanistic studies of initiation remain incredibly challenging. This is primarily because initiation is an extraordinarily dynamic, multi-step process that proceeds through a large number of short-lived intermediate states that are very difficult to observe and characterize using conventional approaches. During initiation, a set of essential initiation factors (IFs) transiently interact with both ribosomal subunits and a specialized initiator tRNA in order to guide their assembly at the start codon of the mRNA to be translated. Although evidence suggests that the IFs, ribosomal subunits, and tRNA undergo functionally important structural rearrangements during this process, very few of these rearrangements have been directly observed and/or characterized, and for those that have, it has been at very low resolution. The long-term goals of this project are to use powerful combinations of reagents and techniques that are uniquely available in our and our collaborators? laboratories to overcome the challenges associated with mechanistic studies of initiation. Specifically, we will use state-of-the-art single-molecule fluorescence microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics of initiation. These studies will be enabled by a new approach that we have developed for introducing fluorophores into ribosomes at positions that are highly desirable, but that have thus far remained out of reach. In Aim 1, we will investigate the mechanism through which bacterial IF2 transiently binds to a ribosomal initiation complex (IC) based on the small, 30S, ribosomal subunit (30S IC); determines whether the 30S IC is carrying an accurately selected initiator tRNA that is properly base-paired to a correctly selected start codon; and, if so, recruits and facilitates joining of the large, 50S, ribosomal subunit to the 30S IC. In Aim 2, we will investigate the mechanism through which bacterial IF3 and IF1 ensure the accuracy with which the start codon is selected during initiation. In Aim 3, we will extend our studies to investigate the mechanism of eukaryotic translation initiation, focusing our attention on eukaryotic-specific aspects of the mechanism through which the eukaryotic homolog of IF2, eukaryotic IF5B (eIF5B), regulates subunit joining during eukaryotic initiation.